1. Field of Endeavor
The present invention relates to sorting, amplification, detection, and identification and more particularly to sorting, amplification, detection, and identification of nucleic acid subsequences in a complex mixture.
2. State of Technology
United States Published Patent Application No. 2005/0032729 by Venkatakrishna Shyamala for identification of oligonucleotides for the capture, detection and quantitation of West Nile virus published Feb. 10, 2005 provides the following state of technology information: “The (Patent Application No. 2005/0032729) invention is based on the development of a sensitive, reliable nucleic acid-based diagnostic test for the detection of WNV in biological samples, particularly blood samples, from potentially infected subjects. The techniques described herein utilize extracted sample nucleic acid as a template for amplification of conserved genomic regions of the WNV sequence using transcription-mediated amplification (TMA), as well as in a 5′ nuclease assay, such as the TaqMan® technique. The methods allow for the detection of as few as 10 copies of the target WNV sequence in viremic samples. Moreover, the methods described herein provide for a one-pot analysis wherein captured sample nucleic acids can be subjected to amplification and detection in the same container. Using the methods of the invention, infected samples can be identified and excluded from the blood supply for transfusion, as well as for the preparation of blood derivatives.”
United States Published Patent Application No. 2005/0042597 for a Viral detection system by Thuy Diem Pham published Feb. 24, 2005 provides the following state of technology information: “RT-PCR based detection systems for avian leukosis/sarcoma virus in unfertilized chicken eggs have been developed. In this assay, the virus can be directly isolated from the egg albumen and the viral RNA efficiently screened by RT-PCR. The amplified RT-PCR product is then directly sequenced, in order to determine avian leukosis/sarcoma virus viral subgroup specificity. Systems specifically designed for effective detection of avian leukosis/sarcoma virus in chicken eggs have been refined, modifications of such systems for use in adult birds are also available. The combined use of RT-PCR and direct sequencing of the RT-PCR product provides a new approach for identifying ALSV-infected poultry. Hence, the present invention makes available molecular-based diagnostic methods for the rapid detection of ALSV retroviruses for use by the poultry industry and public health agencies.”
United States Published Patent Application No. 2005/0233314 by Jyh-Lyh Juang et al for Sensitive and quantitative detection of pathogens by real-time nested PCR published Oct. 20, 2005 provides the following state of technology information: “The (Patent Application No. 2005/0233314) invention provides a method for detecting RNA or DNA pathogens in a sample. The (Patent Application No. 2005/0233314) invention also provides a method for quantifying RNA or DNA pathogens in a sample. Both methods comprise subjecting a sample suspected of containing an RNA or DNA pathogen, to real-time nested PCR. ‘Real-time’ detection allows one to measure the accumulation of amplified product during the course of the reaction, rather than simply analyzing the final product amount following the course of sequential cycles of amplification. ‘Nested’ PCR generally comprises a two-staged polymerase chain reaction process. In a first-stage polymerase chain reaction, a pair of ‘outer’ oligonucleotide primers are used to amplify a first nucleotide sequence. In a second-stage polymerase chain reaction, a second set of ‘inner’ or ‘nested’ oligonucleotide primers are used to amplify a smaller second nucleotide sequence that is contained within the first nucleotide sequence. In the methods of the invention, both stages of nested PCR are based on real-time amplification. The method of the invention is capable of detecting or quantifying less than 10 copies of RNA or DNA in a sample. The method of the invention may be used to detect or quantify SARS-CoV in a sample.”
United States Published Patent Application No. 2006/0134609 by Jeffrey, M. Linnen et al for compositions and methods for determining the presence of SARS coronavirus in a sample published Jun. 22, 2006 provides the following state of technology information: “A novel coronavirus has been identified that causes serious disease in humans. The disease manifests itself with a constellation of clinical findings that have been named the ‘severe acute respiratory syndrome’ or ‘SARS.’ The virus was first identified in China and has shown potential to spread rapidly to other countries. There is no known treatment and there has been a high fatality rate among patients who have presented with pneumonia due to the virus. The signs and symptoms of SARS are common to many diseases. At present, isolation of the patient for periods of 10 days after resolution of disease is recommended to stem the spread of the disease.
The genome of SARS-CoV was recently sequenced and initial diagnostic tests have been developed, including tests to detect antibodies to the virus and polymerase chain reaction (PCR) assays to detect viral sequences. The antibody tests are inadequate because 10-14 days or more are required for antibodies to the virus to develop to detectable levels. The PCR tests initially developed appeared to be highly specific but were sensitive in only about 50% of suspected cases. These PCR tests all amplified a sequence located in the region from about nucleotide 15000 to nucleotide 19000 in the genome.
The low sensitivity of these initial PCR tests may have several causes. For example, the PCR primers may be cross-reacting with other sequences in the samples, thereby resulting in the production of unwanted amplification products. Also, the amount of nucleic acid from SARS-CoV may be below a threshold level of detection or inhibitors in the reaction mixture may be digesting the target nucleic acid or interfering with amplification and/or detection. In addition, because SARS-CoV contains genomic RNA, these initial PCR tests may be performing an inefficient reverse transcription step prior to amplification by PCR. Thus, a need exists for a method which allows for the rapid, sensitive and specific detection of SARS-CoV nucleic acid in a test sample. And for such a method to be of clinical significance, it should be capable of distinguishing the presence of SARS-CoV from that of human coronavirus strains 229E (HCoV-229E) and OC43 (HCoV-OC43), as these latter two viruses are responsible for about 30% of mild upper respiratory tract illnesses.
The (Patent Application No. 2005/0042597) invention relates to oligonucleotides useful for determining the presence of SARS coronavirus in a test sample. The oligonucleotides . . . may be incorporated into detection probes, capture probes and amplification oligonucleotides, or used in various combinations thereof.”